Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

Influence of the amount of antigen and the interval on the secondary reaction

The course of the revaccination reaction in mice immunized with different doses of sheep red blood cells was determined at different intervals after the primary stimulus. The maximum level of haemagglutinating antibodies in the secondary reaction was found after a high primary and secondary antigenic stimulus. On the contrary, if the level of haemolytic antibodies was determined, the higher was the primary antigenic stimulus, the lower was the secondary antibody response. Differences between haemagglutinins and haemolytic antibodies were also manifested in the earlier onset of the maximum haemolytic secondary reaction (five months after the first dose of antigen); the maximum haemagglutination response was not attained until eight months after the primary dose of antigen. The results comfirm that the basis of preparation for the secondary reaction is proliferation of immunologically activated Y cells; differences in the haemolytic and haemagglutination response are related to differences in the character of the antigenic determinants of sheep red cells.     

Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs.

Immunofluorescence, quantitative immunoprecipitation, and inhibition of bacterial agglutination and passive hemagglutination indicate that cross-reactive antigenic determinants are present on the surface of Rhizobium trifolii and clover roots. These determinants are immunochemically unique to this Rhizobium-legume cross-inoculation group. The multivalent lectin trifoliin and antibody to the clover root antigenic determinants bind competitively to two acidic heteropolysaccharides isolated from capsular material of R. Trifolii 0403. The major polysaccharide is an antigen which lacks heptose, 2-keto-3-deoxyoctulosonic acid, and endotoxic lipid A. The minor polysaccharide in the capsular material of R. Trifolii 0403 contains the same antigen in addition to heptose, 2-keto-3-deoxyoctonate, and lipid A. The acidic polysaccharides of two strains of R. trifolii share the clover r-ot cross-reactive antigenic determinant despite other differences in their carbohydrate composition. Studies with monovalent antigen-binding fragments of anti-clover root antibody and Azotobacter vinelandii hybrid transformants carrying the unique antigenic determinant suggest that these polysaccharides bind R. trifolii to the clover root hair tips which contain trifoliin.     

Enzymatic and immunochemical properties of lysozyme—Restriction of recognition of lysozyme antigenic determinants in pigs

Pigs immunized with lysozyme responded by producing only nonprecipitating antibody throughout the immunization period. Fig antilysozyme antibodies were found to be resistant to papain fragmentation, only 33% of the antibodies were fragmented with papain. From the binding of fluorescein labeled or ¹⁴C-labeled lysozyme to antilysozyme antibodies it was concluded that the antibodies elicited in pigs recognized only two antigenic determinants of lysozyme. These results were confirmed from the binding of Fab fragments to ¹⁴C-lysozyme. Fab fragments prepared from precipitating rabbit antilysozyme antibody bound ¹⁴C-lysozyme at a molar ratio of Fab/lysozyme = 3. Therefore nonprecipitating antibodies are the outcome of recognition of only two antigenic determinants on lysozyme and inability to form a lattice structure when antibody and antigen interact. This work emphasizes the limitations of using antibodies as a biological reagent for delineating the antigenic determinants on proteins.     

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.     

Use of monoclonal antibodies for the production of a plague antibody erythrocyte diagnostic agent

Monoclonal antibodies to Yersinia pestis capsular antigen were fixed onto the surface of formulated sheep red blood cells. The preparation thus obtained was compared with commercial antibody erythrocyte diagnosticum in the passive hemagglutination test aimed at the search for the capsular antigen in the suspensions of Yersinia pestis museum cultures and in the antigen neutralization test aimed at the search for antibodies in the sera of wild and laboratory animals having had plague. Monoclonal erythrocyte diagnosticum proved to be suitable for the detection of both the capsular antigen and antibodies. The comparison of the results of the passive hemagglutination test and the enzyme immunoassay demonstrated the presence of very close relationship between them.     

Immunohistochemical distribution of the antigenic determinants detected by monoclonal antibodies to carcinoembryonic antigen

By using four distinct monoclonal antibodies to CEA, the molecular profile of which was clarified in our accompanying companion paper, immunohistochemical distribution of the antigenic determinants on both cancerous and noncancerous tissues as well as fetal tissues was studied with the use of the immunoperoxidase method. All of the monoclonal antibodies recognize different antigenic determinants on the tissue section. None of the antibodies stained granulocytes in the peripheral blood or in the normal liver tissues tested. Three of our monoclonal antibodies stained columnar epithelial cells in morphologically normal colonic mucosa; however, monoclonal antibody YK024 did not stain them. This antibody was also found to be unreactive with intestinal metaplasia lesions of the stomach, but reacted with a 16-wk-old fetal stomach as well as with cancerous parts of the colon and of the stomach. Moreover, it was found that this monoclonal antibody mainly reacted with moderately or poorly differentiated adenocarcinoma lesions of the colon and the stomach. Periodic acid treatment in this study, together with trypsin treatment on the antigen as described in our accompanying companion paper, may suggest that this antibody recognizes the carbohydrate antigenic determinant in nature.     

Localization of antigenic determinants using monoclonal antibodies and alpha-NH2-terminal labeling of polypeptide chains

A method for localization of antigenic determinants in a polypeptide chain of unknown primary structure was proposed. A protein is modified at NH2-terminal and epsilon-NH2-groups of lysine residues with maleic anhydride and then is subjected to partial enzymatic cleavage. Newly formed NH2-terminal groups are tagged with radioiodinated Bolton--Hunter's reagent. The labeled fragments of the antigen are then demaleylated. Comparison of the two longest labeled fragments, only one of which still binds monoclonal antibody, makes it possible to define the location of the antigenic determinant along the polypeptide chain. The method was tested on the bovine tryptophanyl-tRNA synthetase using earlier prepared monoclonal antibodies against this enzyme.     

Polymorphism of murine major histocompatibility Class I antigen: assignment of putative allodeterminants to distinct positions of the amino acid sequence within the first external domain of the antigen

Forty-five new monoclonal antibodies reacting with the mouse H-2Dd antigen have been established. The specificities of 34 of these antibodies were mapped into the first external domain (N) of the Dd antigen by testing reactivities with the products of mosaic H-2 genes in which the coding sequences of the first and/or the second external domains of the H-2Dd genes were recombined in vitro with the remaining portion of the H-2Ld gene. These antibodies reacted with at least 13 distinct allodeterminants located in the N domain, composed of 91 amino acids, as judged from panel tests carried out on various H-2 haplotypes. To assign possible positions of antigenic determinants of these and other anti-H-2Dd antibodies, we compared primary sequences of seven H-2 antigens and searched for correspondence between the pattern of amino acid substitutions in the N domain, allowing 15 positions to be assigned for the antigenic sites. These putative antigenic determinants were assessed for possible relationships with several parameters of protein secondary structure postulated according to predictive methods. Many of these sites appear to be associated with greatest local hydrophilicity, known to correlate with sites of antibody binding in various proteins. We therefore propose that some of the correspondences found in this work represent structural correlates of allodeterminants.     

Blocking of human T lymphocyte functions by anti-Leu-2 and anti-Leu-3 antibodies: differential inhibition of proliferation and suppression

We have shown previously that monoclonal antibodies to the Leu-2 and Leu-3 T cell antigens block the response of their respective subsets in allogeneic MLR. The present study was an effort to explore the mechanism of inhibition and to determine if anti-Leu-2 and anti-Leu-3 antibodies affect the responses to stimuli in addition to alloantigens. Our results indicate that antibodies to Leu-2 and Leu-3 have profound inhibitory effects on proliferation by their respective T cell subsets responding to a variety of stimuli, including specific soluble antigens and alloantigen. This effect was characterized by the following features: a) For optimal inhibition of proliferation, antibody must be present at the onset of antigenic stimulation. b) Inhibition is augmented by increasing the concentration of antibody or decreasing the concentration of antigen. c) Fab fragments of both anti-Leu-2a and anti-Leu-3a antibodies also block proliferation. In addition to their effects on T cell proliferation, anti-Leu-3 antibody blocked T cell-dependent lg synthesis induced in MLR, and anti-Leu-2 antibody prevented the induction, in vitro, of Leu-2+3- suppressor cells of lg synthesis. Taken together, these results suggest that antibodies to antigenic determinants on the Leu-2 and Leu-3 molecules competitively block segments of these structures that bind to alloantigen or nominal antigen. On the other hand, anti-Leu-2a antibody failed to block suppression of the MLR by in vivo activated, antigen-specific Leu-2+3- suppressor cells, which suggests that the Leu-2a epitope does not transmit antigen-specific signals from these differentiated suppressor T cells.     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Schistosoma mansoni: detection and characterization of antigens and antigenemia by inhibition enzyme-linked immunosorbent assay (IELISA)

An inhibition enzyme-linked immunosorbent assay (IELISA) was used to detect the presence of schistosome antigens obtained from cercariae, adult worms, and eggs of the parasite. Using appropriate titers of Schistosoma mansoni infected mouse serum (IMS), it was possible to detect less than 10 ng/ml of schistosome antigen when added to phosphate-buffered saline (PBS, pH 7.2) or normal human serum (NHS). The sensitivity of the test was highly contingent on the number of experimental variables including antibody titer and antigenic source. The results of specificity studies were complicated. Although there was no cross-reactivity detected with other unrelated antigen preparations, extensive cross-reactivity between various schistosome species and "stage-specific" antigens was observed. The IELISA, utilizing IMS, can quantitate the degree of antigenic cross-reactivity, i.e., genus-specific and cross-reacting antigenic determinants. Soluble egg antigen (SEA) preparations obtained from S. mansoni and S. japonicum actually "cross-reacted" more than cercarial- and egg-derived antigens obtained from the same species (S. mansoni). This test also showed a 32-fold increase in specificity for the quantitative detection of specific antigenic determinants when monoclonal antibodies were used to restrict the heterogeneity of the measured response. The technique proved satisfactory for the quantification of parasitic burden in mice and the detection of active infections in humans. Circulating antigen disappeared with a t 1/2 of 72-96 hr after successful treatment.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Determination of the internal association constant as a measure of the affinity of erythocyte-fixed antibodies that interact with mono- and multivalent antigens]

The fraction of antibody-bound determinants of a polyvalent antigen is determined by the fraction of free antigen molecules and the valence of the antigen. The internal association constant for antibodies fixed onto erythrocytes can be found by the suspension method, the index obtained in this process being similar when both monovalent and polyvalent antigens are used. The sensitivity of the antibody-erythrocyte diagnostic preparation in the passive hemagglutinin test depends on the affinity of the active centers of antibodies.     

Mapping antibody binding sites on cytochrome c with synthetic peptides: Are results representative of the antigenic structure of proteins?

Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.     

Development of immunoreagents to ciliary zonules that react with protein components of elastic fiber microfibrils and with elastin-producing cells

We describe the generation of a monoclonal antibody library to ocular zonule components and the characterization of three monoclonal antibodies: 1) one specific for microfibrillar associated glycoprotein (MAGP), a component of both ocular zonules and microfibrils of elastin fibers, 2) an antibody to an as yet unidentified 70,000 dalton antigen that is present in abundance in the extracellular matrix (ECM) of elastin-producing cells, and 3) an antibody reacting with the 67000 dalton subunit of the elastin receptor. The presence of antigenic determinants common to the ocular zonule and elastic fiber microfibrils suggests that zonules, which can be obtained in relatively pure form, can provide a valuable resource for characterizing proteins common to both microfibrillar structures.     

Structural proteins of mammalian RNA tumor viruses: relatedness of the interspecies antigenic determinants of the major internal protein.

The relatedness of antigenic determinants of purified major core proteins of the murine, feline, RD 114/baboon, and woolly monkey/gibbon ape groups of RNA tumor viruses was examined by competition radioimmunoassay. In assay systems of a homologous antigen and antiserum, high affinity competition for binding to all of the antibodies was observed only with the homologous unlabeled protein; the core proteins of other groups of viruses showed only low affinity binding of a small fraction of antibodies, presumably those reactive with the interspecies determinants, at concentrations of competing protein 10- to 100-fold greater than that of the labeled antigen. The cross-reactive (interspecies) antigens of every two viruses were selectively examined by precipitating the purified 125-I-labeled protein with antiserum against each of the other proteins. The extent to which these shared determinants were common to the other viruses was then tested by the effectiveness of the proteins of each virus to compete for antibody binding. Several classes of interspecies determinants were distinguished: those common to two of the groups of viruses, others to three, and some to all four. Moreover, an even greater variety of interspecies determinants was indicated by differences in the affinity of the individual proteins for antibody binding, supporting the hypothesis that there are at least several, if not many, different interspecies determinants with a broad spectrum of antigenic cross-reactivity. These studies suggest that the murine and feline viruses are closely related as they contain cross-reactive antigenic determinants not shared with the other viruses, that the feline virus is more closely related to the woolly monkey virus than to RD 114, and that the RD 114 and woolly monkey viruses retain interspecies determinants shared relatively equally with each of the other viruses.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Interactions between precipitating and nonprecipitating antibodies in the formation of immune complexes

In the present study, we used monoclonal antidinitrophenol (DNP) antibodies to determine certain of the biophysical characteristics of precipitating and nonprecipitating antibodies. In addition, we studied the dynamics of immune complex (IC) formation when precipitating antibodies react with antigen in the presence of nonprecipitating antibodies. The antigen utilized in these studies was DNP-bovine serum albumin. All isolated nonprecipitating anti-DNP antibodies were of the IgG2b isotype, whereas all antibodies with other isotypes (IgG1, IgG3, IgM, IgA and IgE) were precipitating. Nonprecipitating antibodies did not differ significantly from precipitating antibodies in affinity, valence, or isoelectric point. Nonprecipitating antibodies inhibited the formation of precipitable IC between antigen and precipitating antibodies. In addition, preformed IC precipitates were solubilized by nonprecipitating antibodies. The solubilization of IC precipitates was influenced by the isotype of the precipitating antibody and by the antibody:antigen ratio in the IC precipitate. By isokinetic sucrose density centrifugation, we determined that solubilization of IC precipitates by nonprecipitating antibodies was associated with release of free precipitating antibody and formation of soluble IC between the antigen and the nonprecipitating antibody. In conclusion, in this study the nonprecipitating property of mouse anti-DNP antibodies is isotype-specific. Nonprecipitating antibodies compete and displace precipitating antibodies from the antigen, resulting in inhibition of IC precipitation and in IC solubilization. On the basis of the present results, we postulate that antibody-antibody interactions are important determinants of precipitating ability, and that these interactions are a characteristic of antibody isotype.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.